High Density Lipoproteins (HDL, i.e. “good cholesterol”) are protein-heavy particles of lipid and protein which circulate in the blood and play a primary role in removing fatty deposits from arterial walls and returning them to the liver for elimination. As such, high blood concentrations of HDL are associated with reduced formation of arterial plaques, atherosclerosis, and incidence of myocardial infarctions.
Human apolipoprotein-AI (“human Apo-AI”) is the primary protein constituent of HDL. For this reason, Apo-AI and its variants have received considerable interest as potential therapeutic agents for the treatment of atherosclerosis and a number of other acute or chronic disease conditions. Variants of human Apo-AI of potential therapeutic interest include naturally occurring isoforms (e.g. mature protein and pro-protein), naturally occurring point-substitution variants (e.g. Milano and Paris), created point-substitution variants, and peptide mimetics (e.g. fragments of Apo-AI comprising individual secondary structural motifs).
In animal cells, Apo-AI is initially expressed in the endoplasmic reticulum as a 267 amino acid preproprotein. Following cleavage of the 18 amino acid signal peptide and transport across the Golgi apparatus, the proprotein is secreted into the extracellular space. Subsequently, the 6-amino acid N-terminal pro-sequence is cleaved, leaving the 243 residue, mature protein (Proc. Nat. Acad. Sci. 80, 2574 (1983)).
Apo-AI can be purified from serum, but has also been expressed as a recombinant protein in multiple expression systems, including mammalian cell culture, insect cells, plant seed, and bacterial cells. Due to its amphipathic nature, solubility of the purified protein is fairly low: in the range of 1-5 mg/mL. In one example, commercially available human Apo-AI is sold as a solubilized protein solution of 1 mg/mL for delipidated, purified protein (See for example, Catalog No. CYT-037-APOAI1 mg/ml solution contains 10 mM Ammonium Bicarbonate at pH7.4, ProSpec-Tany TechnoGene Ltd, East Brunswick, N.J., USA). Because of this poor solubility, and the need to achieve more concentrated formulations for therapeutic administration, purified Apo-AI has typically been solubilized at higher concentrations in conjunction with various lipid co-solutes. For example, recombinant pro-Apo-AI overexpressed in bacteria was solubilized by addition of dimyristoyl phosphatidylcholine (EP 0308336A2). Other applications have disclosed the use of sphingomyelin or 1-palmitoyl-2-oleoyl phosphatidylcholine to improve the protein's solubility (U.S. Pat. No. 7,435,717). However, these methods only demonstrate actual Apo-AI solubilities in the range of about 20 mg/mL or less.
Because of the relatively poor solubility of Apo-AI under all known conditions, approaches for therapeutic administration of the protein have called for infusion of a large volume of protein solution over a long period of time. For example, in U.S. Pat. No. 7,435,717, methods disclose administering approximately 50 mL solution containing 10 mg/mL Apo-AI, to a patient via infusion over a course of approximately 2 hours
Administration of a single Apo-AI dose would be made considerably more convenient if it could be done via syringe, rather than through infusion of a relatively large volume of liquid for an extended time. This would enable, for example, a patient to self-inject at home in a matter of minutes, rather than having to go to a hospital for a multi-hour infusion. However, since syringe injection would typically be about 1-50, or 1-25 mL or less in volume, and suggested daily or weekly dosages of Apo-AI have typically been on the order of hundreds to thousands of milligrams, this would require a solution containing Apo-AI at a concentration far higher than is possible to attain by any known method to date. However, while there has been considerable effort by pharmaceutical companies to produce new compounds for treating deficiency of HDL diseases, such as, atherosclerosis, there are currently no drugs on the market that have the ability to promote the mobilization and efflux of stored cholesterol from macrophages located in atherosclerotic plaques. Methods for increasing the solubility of Apo-AI to form stable compositions comprising these higher concentrations of Apo-AI above what is presently available, would solve this problem.